Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.Plastids conduct vital biosynthetic functions, and many essential reactions are located exclusively within these organelles. However, plastids contain only limited coding information in their own DNA. Endosymbiotic evolution has resulted in the transfer to the nuclear genome of genes encoding the vast majority of plastid proteins. As a consequence of this displacement of genetic material, plastids had to evolve mechanisms to reimport cytoplasmically synthesized precursor proteins from the cytosol (1-6). In most cases, nucleus-encoded plastid proteins are synthesized in the cytosol as higher molecular weight precursors, with a cleavable N-terminal sequence called transit peptide (7). Transit peptides contain all the information that is necessary and sufficient for import. During the actual import process, transit peptides are proteolytically removed by a stromal processing peptidase. Transit peptides can be simple, as found for stroma proteins, or bipartite, as found for proteins destined to thylakoids. In the latter case, the N-terminal part directs the precursor to the stroma, whereas the non-cleaved C-terminal part directs the partially processed precursors to their final intraorganellar destination; i.e. the thylakoid membranes and the thylakoid lumen. Ultimate precursor maturation occurs by virtue of the thylakoid-processing peptidase (8).During chloroplast import, the transit peptide first recognizes the chloroplast surface in a process involving membrane lipids and the translocon at the outer chloroplast envelope (TOC) 3 (9). The TOC complex consists of three distinct core subunits: the GTP-dependent Toc34 and Toc159 receptors and the translocation channel protein Toc75 (10, 11). Translocation across the inner envelope is mediated by another multiprotein complex, the translocon at the inner chloroplast envelope (TIC) and requires ATP in the st...